Detection of mRNA Expression and Copy Number Variations Within the Goat FecB Gene Associated With Litter Size

The Booroola fecundity (FecB) gene, as the first major fecundity gene identified in Booroola sheep, has attracted careful attention. So far, previous research have uncovered the FecB mutation (Q249R) as the main mutation by virtue of which sheep exhibits multiple lambing phenomena. This mutation is now being intensively studied and widely used. However, such effect of the FecB mutation has not been applied to goats, and similar types of the FecB gene in goats still need to be studied. Thus, the current study attempted to verify potential mutations in the goat FecB gene as well as investigate their functions related to fecundity. First, FecB expression was investigated in six different goat tissues, and we found that FecB expression was highest in the mammary gland, followed by the ovary. Next, the influence of the FecB gene was analyzed from a new perspective, where five potential copy number variations (CNVs) (CNV1–5) within the FecB gene were identified for the first time, and then their effects on litter size were measured. Our results point out that CNV3 (P = 3.44E-4) and CNV5 (P = 0.034) could significantly influence the litter size of goats. Identically, the combination genotype of CNV3 and CNV5 which consisted of their dominant genotypes was also significantly associated with goat litter size (P = 7.80E-5). Hence, CNV3 and CNV5 could serve as potential DNA molecular markers applied to DNA editing and DNA microarray. Additionally, the abovementioned study has laid a theoretical foundation for the detection of potential fertility-related quantitative trait loci within the goat FecB gene.


INTRODUCTION
The Booroola fecundity (Fec B ) gene, also named the bone morphogenetic protein receptor 1B (BMPR1B) gene, was first recognized as a high-prolificacy major gene in Booroola sheep (1) due to the multi-lamb phenomenon contributed by the Fec B mutation (Q249R) (2,3). Moreover, as a receptor of bone morphogenetic proteins (BMPs) (4), it is involved in the regulation of follicle-stimulating hormone (FSH) levels, which regulates the ovulation rate in animals and humans (5,6). In detail, the diminution of the BMP signal reduced by Q249R leads to an increase in folliclestimulating hormone (FSHR) and luteinizing hormone receptor (LHR) density and also reduces apoptosis to increase ovulation (7)(8)(9). Furthermore, the attenuation of BMP signaling can maintain the reserve of primordial follicles while promoting follicular growth and ovulation, which contributes to the overall fertility of a female (10). Subsequently, a previous study showed that the Q249R mutation can regulate the expression of BMP/SMAD signaling and significantly upregulate the expression of fecundity-related genes such as STAR, BMP6, and BMP2 (11). Moreover, it can increase ovulation by suppressing the expression of SMAD6, which acts as an inhibitor of the BMP/SMAD signaling pathway (12).
To date, the Fec B gene has been deeply studied in several breeds and strains of sheep such as Hu, Chinese Merino prolific meat strain, Suffolk, Dorset, and Charolais sheep breeds (13,14), especially the Q249R locus which was successfully applied in sheep breeding as a significant DNA marker. Although only Q249R is regarded as the major mutation related to high fertility in sheep, there are also several identified mutations within the Fec B gene worth studying-for instance, g.29362047T>C and g.29427689G>A were found to be significantly associated with litter size in Hu sheep (15). In addition to single-nucleotide polymorphisms (SNPs), five insertion/deletions (indels) (4, 10, 12, 17, and 23 bp) in the BMPR1B gene were also verified in Chinese Australian White sheep. However, among them, only the 10-bp indel significantly affected the sheep litter size (16). In goat, a previous study has revealed that Smad signaling, steroidogenesis, and cell viability in granulosa cells have been altered upon modulation of the Fec B gene which was similar to that documented in sheep breeds carrying the Fec B mutation (17). Moreover, several research were devoted to the detection of potential mutations in goat Fec B gene, but no major highfertility-related mutation was discovered in goat until now. In detail, C94T was identified to exert a significant influence on the litter size of Liaoning cashmere goats (18). Apart from that, three novel SNPs, including G773C, A775G, and G777A, have also been uncovered in Assam hill and Markhoz goats, respectively, but their specific functions still need to be studied (19,20). Altogether, as a major fecundity-related gene, the Fec B gene as well as its function has attracted careful attention (20)(21)(22)(23)(24)(25), and a number of SNPs and indels within the Fec B gene have also been examined to varying degrees (26)(27)(28). However, almost all studies have focused on sheep, while no major quantitative trait locus (QTL) has yet been identified in goats. Additionally, we found that all relevant research focus on SNPs and indels without paying attention to copy number variation (CNV), which is also one of the promising DNA markers. Therefore, in this work, we aimed to investigate potential CNVs within the BMPR1B gene and, for the first time, measure their effects on goat litter size, thereby trying to provide information for DNA editing and microarray as well as laying the theoretical foundation for improving goat fecundity.

Animals, Total RNA, and Genomic DNA Isolation
In order to obtain RNA samples for further experiments, a total of six different tissues (corpus luteum, large follicle, skeletal muscle, uterus, ovary, and mammary gland) of the goat were collected (n = 3). Total RNA extraction was carried out using TRIzol total RNA extraction reagent (Takara, Dalian, China), following the instructions of the manufacturer. Then, 1% agarose gel electrophoresis in 6× loading buffer was used to evaluate the integrity of total RNA. RNA was then quantified and qualitatively analyzed using a Nanodrop 2000 Spectrophotometer. Qualified RNA samples were stored at −80 • C. To accomplish the further experiment, first-strand cDNA was synthesized by Prime Script TM RT Reagent Kit (Takara, Dalian, China). After that, these cDNAs were conserved at −20 • C for subsequent experiments.
Shaanbei white cashmere (SBWC) female goats (2-3 years) that were healthy and under the same feeding and management conditions were used as the DNA sample in this work. All genomic DNAs were isolated from the ear tissue of 312 female goats according to the protocol of our previous study (29) and then stored at −40 • C. In addition, all litter size and growth trait records of these goats were provided by the staff from farm records.     SYBR Premix Ex Taq (Takara Biotech, Dalian, China), 1 µl of cDNA, 0.5 µl of each primer, and 3 µl of ddH 2 O, using the same protocol as in our previous study (30). GAPDH was used as the reference gene (31). The results were then analyzed by 2 − Ct method (32,33).

DNA Experiment: Genotyping of CNV Within the Fec B Gene
To identify copy number variations (CNVs), we first searched for potential CNVs in the goat Fec B gene in the Animal Omics database (http://animal.nwsuaf.edu.cn) and found five CNV loci. Additionally, the figure of the goat Fec B gene was drawn using Adobe Illustrator 2021 (Adobe, USA) as shown in Figure 1.

Statistical Analyses
The genotype frequencies of the five CNVs in the tested population was calculated, and the haplotypes were constructed via SHEsis online program (http://analysis.bio-x.cn). After that, the genotype distributions of these CNVs in all single lamb and multi-lamb individuals were analyzed using chi-square test (χ 2 ). Besides that, a linear model was used to estimate the effect of fixed factors on goat litter size (28): where Y ijk is the litter size, µ is the overall mean value for each trait, a i is the fixed-factor age, ß j is the fixedfactor genotype, and e ijk is the random error. For CNVs that displayed more than two genotypes, one-way ANOVA was used to assess their relationship with litter size; for <2 group, t-test was performed.

RESULTS
mRNA Expression mRNA expression was determined in the mammary gland, corpus luteum, large follicle, skeletal muscle, ovary, and uterus tissues. Our results demonstrated that Fec B expression was highest in the mammary gland and was 10-fold higher than that in other tissues. To add, the relative expression of the goat Fec B gene in the skeletal muscle, uterus, and ovary varied from 10-to 50-fold changes compared to those in the corpus luteum and large follicle which were used as control (Figure 2).

CNV Detection: Genotype Frequency, LD Analysis, and Haplotype Construction
To make the detected CNVs easier to describe, all of them were named according to their location at the goat chromosome as CNV1-5 ( Table 2). Moreover, CNV2-5 displayed three genotypes ("loss, " "median, " and "gain"), while CNV1 only manifested two genotypes ("median" and "gain") ( Table 3). Furthermore, "gain" was the dominant genotype among all of them.

Association Analysis
To test whether these five CNVs exerted a significant effect on the litter size of goats, we analyzed the association between them and the litter. Our results point out that CNV3 (P = 3.44E-4) and CNV5 (P = 0.034) were significantly associated with goat litter size (Figure 3; Table 5). The dominant genotype for CNV3 was "median, " while the dominant genotype for CNV5 was "gain." Interestingly, the dominant combination genotype M 3 G 5 could also have a significant influence on litter size (P = 7.80E-5) (Figure 4; Table 6). Additionally, the distribution of different genotypes in different litter-type populations significantly differed from each other ( Table 7).

DISCUSSION
The Fec B gene, which serves as a major gene for high fecundity, has been shown to contribute to the multi-lambing phenomena in sheep. Currently, it is gradually becoming clear how the Fec B gene regulates sheep fecundity, while the specific mechanism of the goat Fec B gene is still poorly understood and the first   (A, B/a, b) within the same row differ significantly at P < 0.01/P < 0.05. major QTL still needs to be disclosed. Here we identified the expression of the Fec B gene in six different goat tissues and strikingly found that it had the highest expression in the mammary gland, indicating that the Fec B gene might also function as a lactation regulator of goats. According to a previous study, an increase in litter size stimulates the expression of RFamide-related peptide mRNA, which might contribute to lactational anestrus in rats (34). Additionally, as the litter size increases, milk yield increases (35)(36)(37)(38), and so does the uptake of net daily amino acid by the mammary gland, which is the basis for milk yield and lactoprotein production (39). In short, the above results indicate that the lactation would be mediated by an increase in litter size. Thus, the Fec B gene, which significantly affects litter size, also presumably has a function related to lactation in goats. This might be the reason why the Fec B gene had the highest expression in the mammary gland in goats. To our best knowledge, this is the first time that the Fec B gene has shown such a high expression in the mammary gland, which leads to an indication that it is worth investigating whether the Fec B gene has a lactation-related function, especially in populations with multiple lambs. Then, following the mammary gland, high expression in the ovary indicated that the Fec B gene might be involved in ovarian activity, thereby influencing goat fertility. However, whether the Fec B Genotype (LSM ± SE) P-value  gene could impact goat fecundity has not yet been sufficiently studied (17). Until now, reported studies have found a series of polymorphisms associated with fecundity within the Fec B gene in sheep. However, related research analyzing the association between mutations in the Fec B gene and fertility in goat are relatively scanty-for instance, G773C was identified to be unique in Assam hill goat, while the association between goat fecundity still needed to be studied (19). T242C was uncovered in Barbari, Beetal, Black Bengal, Ganjam, Jhakana, Osmanabadi as well as Sangamneri goat breeds, but the effect of genotypes was non-significant on litter size (40). Moreover, C94T within the goat Fec B gene was revealed to be significantly associated with litter size in Liaoning cashmere goats (18). Interestingly, a related work investigated the Fec B mutation in goat since its significant influence on multiple lambing in sheep. However, the results pointed out that the FecB mutation did not exert the same effect in goat, which led to a hint that novel significant mutations still need discovering (20). Many reports altogether revealed lots of polymorphisms within the Fec B gene associated with fecundity in sheep, while potential mutations still need discovery in goats. No related research also paid attention on the CNV within this gene. In this study, we first identified five potential CNVs in the Fec B gene and measured their effects on litter size in goats. Our results pointed out that the "gain" genotype, rather than another two genotypes, displayed the highest frequency in all five CNVs. Identically, the G 1 G 2 G 3 G 4 G 5 haplotype was also the dominant haplotype with the highest frequency. In addition to the distribution of the five CNVs, we further analyzed their effect on goat litter size, which was the main purpose of this work. The results based on a large experimental population showed that CNV3 and CNV5 exerted significant effects on litter size in goats (P < 0.05), with "median" and "Gain" displaying a superior phenotype, respectively. In addition, the combination genotypes of CNV3 and CNV5 could also have the same effects on goat litter size. Notably, the combined genotype of "median" (CNV3) and "gain" (CNV5) performed superior litter size, which is consistent with the abovementioned results. Thus, CNV3 and CNV5 could serve as effective DNA markers applied to marker-assisted selection breeding, and individuals, especially with combined genotype "M 3 G 5 " should be selected to improve the litter size of goats.
Given that previous investigations revealing intronic variations could influence the interaction between transcription factors and host genes, we hypothesized that CNV3 and CNV5 might influence the combination ability of DNA sequence with transcription factors to indirectly influence the expression of the Fec B gene (41,42). To add, with BMPs acting as the key intraovarian factors regulating ovarian function (43)(44)(45), their biological effects will be mediated after binding to membranebound receptors. Mutations might influence the process BMPs combing with DNA sequence, thereby changing the related functions. However, how the two CNVs within the Fec B gene influence the goat litter size still needs to be studied.
In conclusion, we have identified five CNVs within the Fec B gene in SBWC goat population and found that CNV3 and CNV5 significantly influenced the litter size of goats. Moreover, this is the first report on the effect of CNVs within the Fec B gene on litter size. Despite this, the specific mechanisms require further investigation.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by the International Animal Care and Use Committee of the Northwest A&F University (IACUC-NWAFU; protocol number NWAFAC1008).

AUTHOR CONTRIBUTIONS
YB, XL, and CP came up with idea and revised the manuscript. YB wrote the manuscript and performed the experiments. WF, YK, KW, and YY collected the goat samples and isolated the genomic DNA. YB, YK, and LQ analyzed the data. All authors approved the final version of the manuscript for submission and contributed to the article and approved the submitted version.